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4
Information Related to Biologic Plausibility
The committee reviewed all relevant experimental studies of 2,4-dichlo-
rophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), pi-
cloram, cacodylic acid, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) that
have been published since Update 2006 (IOM, 2007) and has incorporated the
findings, when it was appropriate, into this chapter or into the biologic-plausibil-
ity sections of Chapters 6–9 when they are of consequence for particular health
outcomes. For each substance, this chapter includes a review of toxicokinetic
properties, a brief summary of the toxic outcomes investigated in animal experi-
ments, and a discussion of underlying mechanisms of action as illuminated by
in vitro studies.
To achieve the goals of this chapter more effectively, the current committee
has slightly modified the presentation of toxicologic information used by previous
Veterans and Agent Orange (VAO) committees. The toxicology chapter of each
earlier update presented information about each of the several hundred potentially
relevant toxicologic articles published in the preceding 2 years. In contrast with
the committee’s responsibility to evaluate each potentially relevant epidemiologic
study of the chemicals of interest published in the preceding 2 years, its charge
with respect to the toxicologic literature is to distill experimental toxicologic
findings to judge whether it is biologically plausible to attribute adverse health
outcomes reported in epidemiologic investigations to the chemicals. The current
committee recognized that for most readers of the VAO series the implications of
most toxicologic results reported are not immediately obvious. Therefore, start-
ing with this update, the committee will focus on integrating and interpreting the
toxicologic evidence rather than delineating the entire body of new experimental
findings.
6

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66 VETERANS AND AGENT ORANGE: UPDATE 2008
Establishment of biologic plausibility through laboratory studies strengthens
the evidence of a cause–effect relationship between herbicide exposure and health
effects reported in epidemiologic studies and thus supports the existence of the
less stringent relationship of association, which is the target of this committee’s
charge. Experimental studies of laboratory animals or cultured cells allow obser-
vation of effects of herbicide exposure under highly controlled conditions that
are difficult or impossible to control in epidemiologic studies. Such conditions
include frequency and magnitude of exposure, exposure to other chemicals, pre-
existing health conditions, and genetic differences between people, all of which
can be controlled in a laboratory animal study.
Once a chemical contacts the body, it begins to interact through the processes
of absorption, distribution, metabolism, and excretion. Those four biologic pro-
cesses characterize the disposition of a foreign substance that enters the organ-
ism. Their combination determines the concentration of the compound in the
body and how long each organ is exposed to it and thus influences its toxic or
pharmacologic activity.
Absorption is the entry of the substance into the organism, normally by
uptake into the bloodstream via mucous surfaces, such as the intestinal walls of
the digestive tract during ingestion. Low solubility, chemical instability in the
stomach, and inability to permeate the intestinal wall can all reduce the extent to
which a substance is absorbed after being ingested. The solubility of a chemical
in fat and its hydrophobicity influence the pathways by which it is metabolized
(structurally transformed) and whether it persists in the body or is excreted.
Absorption is a critical determinant of the chemical’s bioavailability, that is, the
fraction of it that reaches the systemic circulation. Other routes of absorption ex-
perienced by free-ranging humans are inhalation (entry via the airways) and der-
mal exposure (entry via the skin). Animal studies may involve additional routes
of exposure that are not ordinarily encountered by humans, such as intravenous
or intraperitoneal injection, in which the chemical is injected into the bloodstream
or abdominal cavity, respectively.
Distribution refers to the travel of a substance from the site of entry to the
tissues and organs where they will have their ultimate effect or be sequestered.
Distribution takes place most commonly via the bloodstream. The term metabo-
lism is used to describe the breaking down that all substances begin to experience
as soon as they enter the body. Metabolism of most foreign substances takes
place in the liver by the action of oxidative enzymes collectively termed cyto-
chrome P450. As metabolism occurs, the initial (parent) chemical is converted
to new chemicals called metabolites. When metabolites are pharmacologically
or toxicologically inert, metabolism deactivates the administered dose of the
parent chemical reducing its effects on the body. Sometimes metabolism may
activate the compound to a metabolite more potent or more toxic than the parent
compound.
Excretion, also referred to as elimination, is the removal of substances or

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INFORMATION RELATED TO BIOLOGIC PLAUSIBILITY
their metabolites from the body, most commonly in urine or feces. Excretion is
often incomplete, and incomplete excretion results in the accumulation of foreign
substances that can adversely affect function.
The routes and rates of absorption, distribution, metabolism, and excretion
of a toxic substance collectively are termed toxicokinetics (or pharmacokinetics).
Those processes determine the amount of a particular substance or metabolite that
reaches specific organs or cells and that persists in the body. Understanding the
toxicokinetics of a chemical is important for valid reconstruction of exposure of
humans and for assessing the risk of effects of a chemical. The principles involved
in toxicokinetics are similar among chemicals, although the degree to which
different processes influence the distribution depends on the structure and other
inherent properties of the chemicals. Thus, the lipophilicity or hydrophobicity of
a chemical and its structure influence the pathways by which it is metabolized
and whether it persists in the body or is excreted. The degree to which different
toxicokinetic processes influence the toxic potential of a chemical depends on
metabolic pathways, which often differ among species. For that reason, attempts
at extrapolation from experimental animal studies to human exposures must be
done with extreme care.
Many chemicals were used by the US armed forces in Vietnam. The nature
of the substances themselves was discussed in more detail in Chapter 6 of the
original VAO report (IOM, 1994). Four herbicides documented in military records
were of particular concern and are examined here: 2,4-D, 2,4,5-T, 4-amino-3,5,6-
trichloropicolinic acid (picloram), and cacodylic acid (dimethyl arsenic acid,
DMA). This chapter also examines 2,3,7,8-tetrachlorodibenzo-p-dioxin (referred
to in this report as TCDD to represent a single, and the most toxic, congener
of the tetrachlorodibenzo-p-dioxins [tetraCDDs], also commonly referred to as
dioxin), a contaminant of 2,4,5-T, because its potential toxicity is of concern;
considerably more information is available on TCDD than on the herbicides.
Other contaminants present in 2,4-D and 2,4,5-T are of less concern. Except as
noted, the laboratory studies of the chemicals of concern used pure compounds or
formulations; the epidemiologic studies discussed in later chapters often tracked
exposures to mixtures.
TCDD
Chemistry
TCDD is a polychlorinated dibenzo-p-dioxin that has a triple-ring structure
consisting of two benzene rings connected by an oxygenated ring (Figure 4-1);
chlorine atoms are attached at the 2, 3, 7, and 8 positions of the benzene rings.
The chemical properties of TCDD include a molecular weight of 322, a melting
point of 305–306°C, a boiling point of 445.5°C, and a log octanol–water parti-
tion coefficient of 6.8 (NTP substance profile). It is virtually insoluble in water

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68 VETERANS AND AGENT ORANGE: UPDATE 2008
Cl O Cl
Cl Cl
O
2,3,7,8-tetrachlorodibenzo-p -dioxin
FIGURE 4-1 Chemical structure of TCDD.
Figure 4-1.eps
(19.3 ng/L), but is soluble in organic solvents, such as benzene and acetone. It
has been suggested (EPA 2004 Draft Document) that volatilization of dioxin
from water may be an important mechanism of transfer from the aqueous to the
atmospheric phase.
Absorption, Distribution, Metabolism, and Elimination
The absorption, distribution, metabolism, and elimination of TCDD have
been extensively studied in a number of animal models in the last 25 years.
Given the plethora of data, this section only highlights and summarizes key find-
ings. A more exhaustive review may be found at http://www.epa.gov/ncea/pdfs/
dioxin/nas-review.
TCDD is absorbed into the body rapidly but is eliminated slowly. Because
of the slow elimination, the concentration of TCDD in lipid or blood is thought
to be in dynamic equilibrium with that in other tissue compartments and is thus
considered to be reasonable for use in estimating total body burdens. Exposure
of humans to TCDD is thought to occur primarily via the mouth, skin, and lungs.
In laboratory animals, oral administration of TCDD has been shown to result in
absorption of 50–93% of the administered dose (Nolan et al., 1979; Rose et al.,
1976). Similarly, a study performed in a 42-year-old man found that 87% of the
oral dose was absorbed. Dermal absorption appears to be dose-dependent, with
lower absorption occurring at higher doses (Banks and Birnbaum, 1991). Studies
performed in humans indicate that human skin may be more resistant to absorp-
tion (Weber, 1991).
After ingestion and gastrointestinal absorption, TCDD associates primarily
with the lipoprotein fraction of the blood and later partitions into the cellular
membranes and tissues (Henderson and Patterson, 1988). TCDD is distributed to
all compartments of the body; the amounts differ from organ to organ, but most
studies indicate that the primary disposition of TCDD is in the liver and adipose
tissues. For example, in a human volunteer, it was found that at 135 days after
ingestion, 90% of TCDD was in fat (Poiger and Schlatter, 1986); in the rhesus
monkey, TCDD is very persistent in adipose tissue (Bowman et al., 1989). The

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INFORMATION RELATED TO BIOLOGIC PLAUSIBILITY
disposition and elimination of TCDD depend on the tissue examined, the time
that has elapsed since exposure, total exposure, and other factors. For example,
the concentration of cytochrome P450 1A2 (CYP1A2) (Poland et al., 1989) in the
liver is increased by TCDD. Direct binding of TCDD to the CYP1A2 is thought
to result in sequestration of TCDD in the liver and to inhibit its distribution to
other tissues. The importance of CYP1A2 concentrations for the toxic actions of
TCDD has also been shown in studies performed in laboratory animals in which
maternal hepatic CYP1A2 was found to sequester TCDD and protect the fetus
against TCDD-induced teratogenesis (Dragin et al., 2006). In addition, distribu-
tion of TCDD is age-dependent, as shown by studies in which young animals
displayed the highest concentration of TCDD in the liver and older animals the
highest concentrations in kidney, skin, and muscle (Pegram et al., 1995). Finally,
the elimination rate of TCDD, in particular after low exposures, depends heavily
on the amount of adipose tissue mass (Aylward et al., 2005; Emond et al., 2005,
2006).
In laboratory animals and humans, metabolism of TCDD occurs slowly. It
is eliminated primarily in feces as both the parent compound and its more polar
metabolites. However, elimination appears to be dose-dependent; at low doses,
about 35% of the administered dose of TCDD was detected in the feces; at higher
doses, about 46% was observed (Diliberto et al., 2001). The dose-dependent
occurrence of TCDD metabolites in the feces is thought to be due to increased
expression of metabolizing enzymes at higher doses. A measure of elimination
is half-life, which is defined as the time required for the plasma concentration or
the amount of a chemical in the body to be reduced by one-half. The half-life of
TCDD in humans varies with body mass index, age, sex, and concentration and
has been found to vary from 0.4 to over 10 years (Table 4-1).
In light of the variables discussed above and the effect of differences in
physiologic states and metabolic processes, which can affect the mobilization
of lipids and possibly of compounds stored in them, complex models known as
physiologically based pharmacokinetic models have been developed to integrate
exposure dose with organ mass, blood flow, metabolism, and lipid content to
predict the movement of toxicants into and out of each organ. A number of recent
modeling studies have been performed in an effort to understand the relevance
of animal experimental studies to exposures that occur in human populations
(Aylward et al., 2005a,b; Emond et al., 2005).
Toxicity Profile
The administration of TCDD to laboratory animals affects many tissues and
organs. The effects of TCDD in laboratory animals have been observed in a
number of species (rats, mice, guinea pigs, hamsters, monkeys, cows, and rabbits)
after the administration of a variety of doses and after periods that represent acute
(less than 24 hr), subchronic (more than 1 day up to 3 months), and chronic (more

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INFORMATION RELATED TO BIOLOGIC PLAUSIBILITY
than 3 months) exposures. Some differences are observed in the different species,
particularly with respect to their degree of sensitivity, but in general the effects
observed are qualitatively similar. Relatively high exposures of TCDD affect a
variety of organs and result in organ dysfunction and death. The specific organ
dysfunction that constitutes the lethal event, however, is not known. A character-
istic of TCDD exposure is a wasting syndrome with loss of adipose and muscle
tissues and severe weight loss. In most rodents, exposure to TCDD affects the
liver, as indicated by hepatic enlargement, the presence of hepatic lesions, and
impaired hepatic function. The thymus is also sensitive. Finally, in both humans
and nonhuman primates, TCDD exposure results in chloracne and associated
dermatologic changes. As will be discussed in more detail in Chapters 6–9, stud-
ies performed in animal models have indicated that exposure to TCDD adversely
affects the heart, the skin, and the immune, endocrine, and reproductive systems,
and increases the incidence of cancers of the liver, skin, thyroid, adrenal cortex,
hard palate, nasal turbinates, tongue, and respiratory and lymphatic systems (Huff
et al., 1994). When TCDD has been administered to pregnant animals, such birth
defects as cleft palate, malformations of the reproductive organs of the male
and female progeny, and abnormalities in the cardiovascular system have been
observed.
The administration of TCDD to laboratory animals and cultured cells affects
enzymes, hormones, and receptors. In addition to adversely affecting the ability
of specific organs to fulfill their normal physiologic roles, TCDD has been found
to alter the function and expression of essential proteins. Some of the proteins
are enzymes, specialized proteins that increase the rates of chemical reactions
and aid in the body’s ability to convert chemicals into different molecules. The
metabolism of foreign chemicals often changes their biologic properties and in
some cases increases the body’s ability to eliminate them in urine. The enzymes
that are most affected by TCDD are ones that act on or metabolize xenobiotics
and hormones. Xenobiotics are chemicals that are not expected to be present in
the body, and hormones are made by the body and serve as chemical messengers
that transport a signal from one cell to another. Among the enzymes affected by
TCDD, the best studied is CYP1A1, which metabolizes xenobiotics. In labora-
tory animals, exposure to TCDD commonly results in an increase in the CYP1A1
present in most tissues; CYP1A1 therefore is often used as a marker of TCDD
exposure.
Other enzymes that are affected by TCDD are ones that metabolize hormones
such as thyroid hormones, retinoic acid, testosterone, estrogens, and adrenal ste-
roids. Those hormones transmit their signals by interacting with specific proteins
called receptors and in this manner initiate a chain of events in many tissues of
the body. For example, binding of the primary female sex hormone, estrogen, to
the estrogen receptor promotes the formation of breasts and the thickening of the
endometrium and regulates the menstrual cycle. Exposure to TCDD can increase

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2 VETERANS AND AGENT ORANGE: UPDATE 2008
the metabolism of estrogen, and this leads to a decrease in the amount of estrogen
available for binding and activating the estrogen receptor. The ultimate effect of
TCDD is an interference with all the bodily functions that are regulated by estro-
gens. Similarly, the actions of TCDD on the adrenal steroids can adversely affect
their ability to regulate glucose tolerance, insulin sensitivity, lipid metabolism,
obesity, vascular function, and cardiac remodeling. In addition to changing the
amount of hormone present, TCDD has been found to interfere with the ability of
receptors to fulfill their role in transmitting hormone signals. Animal models have
shown that exposure to TCDD can increase the amounts of enzymes in the body
and interfere with the ability of hormones to activate their specific hormone re-
ceptors. Those actions of TCDD on enzymes and hormone receptors are thought
to underlie, in part, observed developmental and reproductive effects and cancers
that are hormone-responsive.
TCDD alters the paths of cellular differentiation. Research performed primarily
in cultured cells has shown that TCDD can affect the ability of cells to undergo
such processes as proliferation, differentiation, and apoptosis. During the pro-
liferative process, cells grow and divide. When cells are differentiating, they
are undergoing a change from less specialized to more specialized. Cellular dif-
ferentiation is essential for an organism to mature from a fetal to an adult state.
In the adult, proper differentiation is required for normal functions of the body,
for example, in maintaining a normally responsive immune system. Processes of
controlled cell death, such as apoptosis, are similarly important during develop-
ment of the fetus and are necessary for normal physiologic functions in the adult.
Apoptosis is a way for the body to eliminate damaged or unnecessary cells. The
ability of a cell to undergo proliferation, differentiation, and apoptosis is tightly
controlled by an intricate network of signaling molecules that allows the body
to maintain the appropriate size and number of all the specialized cells that form
the fabric of complex tissues and organs. Disruption of that network that alters
the delicate balance of cell fate can have severe consequences, including impair-
ment of the function of the organ because of the absence of specialized cells.
Alternatively, the presence of an excess of some kinds of cells can result in the
formation and development of tumors. Thus, the ability of TCDD to disrupt the
normal course of a specific cell to proliferate, differentiate, or undergo apoptosis
is thought to underlie (at least in part) its adverse effects on the immune system
and the developing fetus and its ability to promote the formation of certain
cancers.
Definition of Dioxin-like Compounds and TEF and TEQ Terminology
Many compounds have dioxin-like properties: they have similar chemical
structure, have similar physiochemical properties, and cause a common battery
of toxic responses. Because of their hydrophobic nature and resistance to me-

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INFORMATION RELATED TO BIOLOGIC PLAUSIBILITY
tabolism, these chemicals persist and bioaccumulate in fatty tissues of animals
and humans. Several hundred chemicals—such as the polychlorinated dibenzo-
p-dioxins, polychlorinated dibenzofurans, polybrominated dibenzo-p-dioxins,
polybrominated dibenzofurans, and polychlorinated biphenyls—are described
as dioxin-like compounds (DLCs), although only a few of them are thought to
display dioxin-like toxicity. For most purposes, only 17 polychlorinated dibenzo-
p-dioxins and polychlorinated dibenxofurans and a few of the coplanar poly-
chlorinated biphenyls that are often encountered in environmental samples are
recognized as being true DLCs. In the context of risk assessment, these polychlo-
rinated dibenzo-p-dioxins, polychlorinated dibenxofurans, and polychlorinated
biphenyls are commonly found as complex mixtures when detected in environ-
mental media and biologic tissues or when measured as environmental releases
from specific sources. That complicates the human health risk assessment that
may be associated with exposures to varied mixtures of DLCs. To address the
problem, the concept of toxic equivalency has been elaborated by the scientific
community, and the toxic equivalency factor (TEF) has been developed and in-
troduced to facilitate risk assessment of exposure to those chemical mixtures. On
the most basic level, TEFs compare the potential toxicity of each DLC found in
a mixture with the toxicity of TCDD, the most toxic member of the group. The
procedure involves assigning individual TEFs to the DLCs with consideration of
chemical structure, persistence, and resistance to metabolism. TEF ascribe spe-
cific order-of-magnitude toxicity to each DLC relative to that of TCDD, which
is assigned a TEF of 1.0. The DLCs have TEFs ranging from 0.00001 to 1.0.
When several compounds are present in a mixture, the toxicity of the mixture
is estimated by multiplying the TEF of each DLC in the mixture by its mass
concentration and summing the products to yield the TCDD toxicity equivalent
quotient (TEQ) of the mixture.
Mechanism of Action
TCDD binds and activates the aryl hydrocarbon receptor (AHR). The AHR is
a member of a family of basic-helix-loop-helix (bHLH) transcription factors, that
is, one of many proteins in the cell that controls the transfer (or transcription) of
genetic information from DNA to RNA. bHLH proteins are characterized by the
presence of a string of basic amino acid residues followed by two alpha helices
joined by a loop. Generally, the larger of the two helices participates in binding
to DNA in a specific sequence motif; the specificity is determined largely by the
amino acid sequence of the helix. bHLH transcription factors are dimeric, form-
ing functional heterodimers with other members of the family. By mechanisms
that are poorly understood, binding of the heterodimeric complex to DNA recruits
the transcriptional machinery needed to activate gene expression and results in a
large increase in the rate of synthesis of mRNA molecules for the genes regulated
by the complex and ultimately in a large increase in the corresponding protein.

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4 VETERANS AND AGENT ORANGE: UPDATE 2008
The best known AHR target is the expression of a mixed-function oxidase en -
zyme that was termed aryl hydrocarbon hydroxylase (AHH) in the 1960s and is
now better known as the CYP1A1 enzyme. Expression of this protein is an acute
outcome of AHR activation and may not faithfully represent the consequences of
chronic exposure to AHR ligands.
In its inactive state, the AHR is found in the cytosol of the cell, where it is
protected from proteolytic degradation by several chaperones and cochaperones.
As a receptor, the AHR is a protein capable of receiving and forming a com-
plex with specific substances, termed ligands, which confer on it the ability to
perform a biologic function. In the case of the AHR, the function is to induce
the transcription of specific target genes. Hence, the AHR belongs to a class of
ligand-activated transcription factors. If the ligand is a chemical, such as TCDD,
the AHR dissociates from the chaperones and translocates into the nucleus of the
cell, where it forms a heterodimer with another bHLH protein, the AHR nuclear
translocator (ARNT). This heterodimer binds to its cognate DNA motifs and
recruits the macromolecular complexes needed to initiate gene transcription.
AHR Functional Domains
The AHR contains several regions, or domains, that perform distinct func-
tions. The receptor is a member of the Per-Arnt-Sim (PAS) bHLH subfamily
(Burbach et al., 1992; Fukunaga et al., 1995). The bHLH motif is found in the
amino terminus of the protein and is common to all transcription factors in this
subfamily (Jones, 2004). The members of the bHLH family also have several
highly conserved domains with functionally distinctive biochemical roles. One
of the domains is the basic region, described earlier, which is involved in the
binding of AHR/ARNT complexes to DNA. Another domain is the HLH region,
which facilitates the stable interaction between AHR and ARNT. A third domain
is termed the PAS domain and consists of a stretch of 200–350 amino acids with
high sequence relatedness to protein domains that were originally found in the
Drosophila melanogaster genes period (Per) and single minded (Sim) and in the
AHR’s dimerization partner ARNT; hence the name PAS. The AHR contains two
PAS domains, PAS-A and PAS-B (Ema et al., 1992). The PAS domains support
secondary interactions with other PAS-domain–containing proteins, with the
chaperones and cochaperones, and with many other transcription factors, coacti-
vators and corepressors. The ligand-binding site of AHR is in the PAS-B domain
(Coumailleau et al., 1995) and contains several conserved residues critical for
ligand binding (Goryo et al., 2007). A fourth important domain in the carboxyl
terminus of the protein, is rich in glutamine and is involved in coregulator recruit-
ment and transactivation (Kumar et al., 2001).

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INFORMATION RELATED TO BIOLOGIC PLAUSIBILITY
AHR Ligands
From an environmental point of view, there are two classes of AHR ligands—
synthetic and naturally occurring—that total more than 400 known ligands. Many
of the first ligands to be discovered were synthetic polycyclic aromatic hydrocar-
bons (PAHs), such as 3-methylcholanthrene, benzo[a]pyrene (B[a]P), benzanthra-
cene, and naphthoflavone. The biologic consequences of experimental exposure
of mice to those chemicals led to the prediction of a receptor-dependent mecha-
nism long before the existence of the AHR was directly demonstrated. Compari-
son of the effects of 3-methylcholanthrene treatment in two inbred mouse strains
revealed a major difference in PAH responsiveness. Hepatic CYP1A1 enzyme
increased more than 6-fold after 3-methylcholanthrene treatment in C57BL/6
mice but not in DB/2 mice. Appropriate genetic crosses between responsive
C57BL/6 mice but in non-responsive DB/2 mice indicated that responsiveness
in these prototype strains was inherited as a simple autosomal dominant trait.
The genetic locus defined in the crosses was termed the aromatic hydrocarbon
responsieness (Ahr) locus (Nebert et al., 1982). Molecular biologic studies dur-
ing the next decade showed that responsive and nonresponsive mice had equally
functional CYP1A1 enzymes and that the Ahr locus encoded a regulatory gene
responsible for induction of the Cyp1a1 gene. The members of the polyhalo-
genated aromatic hydrocarbons—such as the dibenzodioxins, dibenzofurans,
and polychlorinated and polybrominated biphenyls—were recognized as AHR
ligands much later, after the discovery by Poland and co-workers that dioxin was
a potent inducer of hepatic AHH in the rat. At that time, it was found that high
concentrations of TCDD could induce AHH activity in the nonresponsive DB/2
mouse to levels as high as those in the responsive C57BL/6 mouse. The difference
between responsive and nonresponsive strains was in sensitivity to the inducer:
DB/2 mice required 18 times more TCDD than C57BL/6 mice for 50% of the
maximal response. Later, a receptor protein for TCDD, 3-methylcholanthrene and
other PAHs, was identified, characterized in the hepatic cytosol of C57BL/6 mice,
and termed Ah receptor (Poland et al., 1976). The available evidence indicated
that the protein was the product of the Ahr locus, which was localized to mouse
chromosome 12 and human chromosome 7, and later cloned (Burbach et al.,
1992; Ema et al., 1992).
Recent work has focused on naturally occurring compounds in hopes of
identifying an endogenous ligand (Denison and Nagy, 2003). Several such natu-
rally occurring compounds have been identified as AHR ligands, including the
tryptophan derivatives indigo and indirubin (Adachhi et al., 2001), the tetrapyr-
roles bilirubin (Sinal and Bend, 1997), the arachidonic acid metabolites lipoxin
A4 and prostaglandin G (Seidel et al., 2001), modified low-density lipoprotein
(McMillan and Bradfield, 2007), several dietary carotenoids (Denison and Negy,
2003; Probst et al., 1993), and cAMP (Oesch-Bartlomowicz et al., 2005). One
assumption made in the search for an endogenous ligand is that the ligand will

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